长效重组人凝血因子Ⅷ研究现状及进展

重组凝血因子Ⅷ(rFⅧ)替代疗法是目前临床上治疗A型血友病最为有效的方法。过去十几年的临床试验已经验证了rFⅧ替代疗法的安全性和有效性,但由于FⅧ的半衰期较短,患者需要隔天或者每周3次进行静脉注射治疗。长期频繁静脉注射治疗不仅给患者带来肉体上的痛苦,还容易产生FⅧ抗体,严重影响治疗效果。以下就目前长效型重组凝血因子Ⅷ的研究进展及存在的主要问题进行了综述。     

凝血因子Ⅷ研究和应用的现状与前景

基因重组型凝血因子Ⅷ（rFⅧ)代替血浆来源的FⅧ（dFⅧ)治疗血友病甲是血友病治疗史上的一次革命,但仍存在难以令人完全满意的地方,而目前研制的二代rFⅧ即B区缺失型FⅧ具有的优点弥补了野生型rFⅧ的不足,必将促使患的选择由pdFⅧ彻底转向rFⅧ。     

因子Ⅷ基因突变的研究进展

凝血因子Ⅷ（FⅧ）是内源性凝血系统中一重要的辅助因子,由于基因缺陷而引起的Ａ型血友病是一种常见的遗传性出血性疾病.近几年来,由于FⅧ基因的阐明及成功的表达,从而使其基因突变的研究得以深入而广泛地展开.文章对这方面最新的研究进展及其采用的新技术作了较全面的介绍,这是迄今遗传性疾病基因缺陷研究中最深入、最完整的一个范例.     

凝血因子Ⅶ基因表达载体的构建及其在BHK细胞中的表达

凝血因子Ⅶ是凝血块形成的起始关键分子之一,它对外源性凝血途径的启动具有重要的作用.为表达具有促凝活性的重组人凝血因子Ⅶ,通过PCR的方法从质粒pUC-FⅦ中扩增人凝血因子ⅦcDNA,并将其定向克隆入真核表达载体pIRESneo中,构建重组表达载体pIRES-FⅦ,测序正确后用脂质体介导的方法转染BHK-21细胞,经G418加压筛选、细胞有限稀释等方法获得克隆细胞株,收集无血清培养上清,进行SDS-PAGE、Westem blot和活性鉴定.结果成功构建了重组表达载体pIRES-FⅦ,实现了其在BHK-21细胞中的表达,且表达产物具有促凝活性.重组人凝血因子Ⅶ在哺乳动物细胞中的成功表达,为整体止血剂的研究奠定了基础.     

重组质粒在减毒沙门氏菌中的稳定性及其对细菌侵袭力的影响

将含有外源基因的重组真核表达质粒pcDNA3-F和pCI-F转化减毒鼠伤寒门氏菌,探讨质粒类型和插入片段对重组质粒在细菌内的稳定性和细菌侵袭力的影响。结果表明,外源质粒可降低减毒沙门氏菌在体外的增殖能力和侵袭力,也影响细菌在鸡体内的存活力;就质粒类型而言,pCI的影响大于pcDNA3,而以携带外源基因的重组质粒影响较为显著;外源基因插入也影响质粒在宿主菌内的稳定性。提示利用减毒鼠伤寒沙门氏菌为载体传递DNA疫苗研究时,要考虑质粒类型与其在宿主菌内稳定性的关系、携带外源基因重组质粒对载体菌侵袭力和存活力的影响等问题。     

凝血因子VII基因表达载体的构建及其在BHK细胞中的表达

凝血因子Ⅶ是凝血块形成的起始关键分子之一,它对外源性凝血途径的启动具有重要的作用。为表达具有促凝活性的重组人凝血因子Ⅶ,通过PCR的方法从质粒pUC-FⅦ中扩增人凝血因子ⅦcDNA,并将其定向克隆入真核表达载体pIRESneo中,构建重组表达载体pIRES-FⅦ,测序正确后用脂质体介导的方法转染BHK-21细胞,经G418加压筛选、细胞有限稀释等方法获得克隆细胞株,收集无皿清培养上清,进行SDS-PAGE、Western blot和活性鉴定。结果成功构建了重组表达载体pIRES-FⅦ,实现了其在BHK-21细胞中的表达,且表达产物具有促凝活性。重组人凝血因子Ⅶ在哺乳动物细胞中的成功表达,为整体止血剂的研究奠定了基础。     

凝血因子Ⅶ及其重组表达新进展

凝血因子Ⅶ是一种维生素K依赖型的单链糖蛋白,在凝血过程中发挥着极其重要的作用,在临床上有广泛的应用,可用于伴有抑制物的血友病、先天性FⅦ缺乏症、血小板无力症及外科手术或严重外伤导致的创伤出血等止血用途.基因重组技术提供了能够大规模制备人凝血因子Ⅶ的有效途径,近年来已尝试并建立了多种人凝血因子Ⅶ的重组表达系统.对重组人凝...     

克隆凝血因子Ⅷ

生物技术业最富雄心且令人振奋的目标之一已经达到。在本期发表的四篇论文中,Genentech公司(在伦敦皇家自由医院Tuddenham等帮助下)和遗传研究所公司(在罗彻斯特Mayo诊所帮助下)报告了指导合成人凝血因子Ⅷ的:C的DNA的克隆,以及克隆DNA在哺乳动物细胞系中表达以生产这种凝血因子的情况。     

IFITM1基因真核表达质粒的构建及鉴定

目的:获得IFITM1基因片段并构建真核表达质粒.方法:采用RT-PCR技术扩增IFITM1,将扩增产物连接至pcDNA3.1载体,对重组质粒进行测序验证,结果:构建了真核表达质粒pcDNA3.1-IFITM1,通过酶切、测序等方法验证完全正确.结论:成功构建了IFITM1基因的真核表达质粒,为下一步探讨IFITM1基因在子宫颈癌HeLa细胞中的作用提供了实验基础.     

pcDNA3.1(+)-CTGF真核表达质粒构建及其在人成骨样细胞SaOS-2细胞中的表达*

目的:构建结缔组织生长因子(CTGF)的pcDNA3.1(+)真核表达质粒(pcDNA3.1(+)-CTGF),并检测其在人成骨样细胞SaOS-2中的表达,为进一步研究CTGF基因在骨发育和骨修复中的机制提供技术支撑。方法:采用PCR方法体外克隆CTGF基因全序列,将其用同源重组技术连接到线性pcDNA3.1(+)载体上,构建pcDNA3.1(+)-CTGF真核表达质粒,并对该质粒进行测序鉴定;鉴定无误后转染至SaOS-2细胞中,观察其48 h的表达情况。结果:基因测序证实pcDNA3.1(+)-CTGF真核表达重组质粒构建成功,与对照组相比,转染SaOS-2细胞48 h后的CTGF表达水平显著上调,达到对照组的4.8×10⁵倍(P＜0.01)。结论:成功构建了pcDNA3.1(+)-CTGF真核表达质粒,并能在人成骨样细胞SaOS-2中稳定表达,为深入研究CTGF基因对骨生成的调控机制奠定了基础。     

小鼠pEGFP-Hoxa11真核表达载体的构建及表达

目的 构建和鉴定Hoxa11和EGFP双基因共表达真核载体.方法 采用DNA重组技术,将目的 基因Hoxa11克隆至含有报告基因EGFP的pEGFP-N1真核表达载体中,构建的真核表达载体pEGFP-Hoxa11经PCR,双酶切及基因测序鉴定;转染至CHO细胞,荧光显微镜下观察重组质粒的表达,提取细胞蛋白Western印迹检测蛋白表达.结果 pEGFP-Hoxa11重组质粒构建成功.构建的真核表达载体pEGFP-Hoxa11能在CHO细胞中有效表达.结论 成功构建了共表达Hoxa11和EGFP的真核表达载体,并能在CHO细胞中有效表达.为进一步研究Hoxa11的功能提供实验基础.     

Construction of a recombinant eukaryotic expression plasmid containing human PDLIM2 gene and its biological activity

In order to obtain a full-length expression plasmid for human PDLIM2 gene, fragment amplification was used to clone its full-length coding sequence (CDS) region. The amplified PCR product was then digested and inserted into the pMD 18-T vector and subcloned into the pIRES2-EGFP plasmid to form the pIRES2-EGFP-PDLIM2 eukaryotic expression vector. After it was transfected to the bladder cancer cell line, BIU-87, the biological activities of high expression were verified by RT-PCR and Western blotting. Meanwhile the mRNA and protein expressions of p65 were detected. Finally we analyzed the effect of overexpressed PDLIM2 on BIU-87 cell proliferation. In conclusion, a recombinant eukaryotic expression vector pIRES2-EGFP-PDLIM2 containing the complete CDS region of PDLIM2 was successfully constructed. PDLIM2 negatively regulated p65 expression and inhibited BIU-87 cell proliferation. We laid the foundations for further research into the function of the PDLIM2 gene in bladder cancer.     

Construction of arsenic-resistant Thiobacillus ferrooxidans recombinant plasmids and the expression of autotrophic plasmid genes in a heterotrophic cell-free system

Recombinant plasmids between Thiobacillus ferrooxidans and Escherichia coli which contain arsenic and antibiotic resistance genes and unique restriction sites have been constructed. The expression of autotrophic T. ferrooxidans genes in a heterotrophic E. coli cell-free system was demonstrated.     

Enhancement of plasmid stability and enzymatic expression by immobilising recombinant Saccharomyces cerevisiae

Immobilisation of cells in a calcium alginate gel improved plasmid stability (up to 50%) and enzymatic expression (up to 57%) of a recombinant Saccharomyces cerevisiaeover-expressing the homologous gene EXG1. The rate of segregational loss in the free cells was 14-fold higher than that of the immobilised cells. Recombinant protein synthesis requires reduced cofactors, which affect the redox balance of the cell.     

一种新型抗菌肽Cecropin B真核表达载体的构建

通过重叠区扩增法人工设计和合成一种新型抗菌肽Cecropin B基因,按正确的阅读框架定向克隆至巴斯德毕赤酵母的高效表达载体pPIC9K上。经PCR鉴定及序列分析,所转化的大肠杆菌DH5ct菌落中含有插入Cecropin B基因的重组质粒pPIC9K—CB,表明成功构建了新型抗菌肽Cecropin B表达载体pPIC9K—CB。     

重组质粒pUDK-HGF 的中试纯化工艺

pUDK-HGF是携带人肝细胞生长因子的裸质粒,目前已进入I期临床试验,因此需要大量符合药学规格的质粒DNA。文中建立了pUDK-HGF中试规模纯化制备的新工艺。流程包括：发酵、离心收获菌体、碱裂解、超滤浓缩碱裂解液、Sephacryl S-1000层析除去RNA并更换缓冲液、plasmidselect捕获超螺旋质粒DNA、琼脂糖凝胶6BFF除盐。新工艺可获得浓度为2.0 mg/mL、纯度在1.70以上的裸质粒原液,符合相关质量标准,并避免使用动物源性的酶及有毒试剂。     

Kinetics of loss of a recombinant plasmid in Bacillus subtilis

Recombinant plasmid pCEDS is structurally unstable in Bacillus subtilis cultures. We have previously shown that stability can be independently increased by changing from a complex medium supporting high growth rates to a chemically-defined medium supporting a lower growth rate and removal of a 4.77-kb EcoRI fragment from pCED3 to give plasmid YS1. Further stabilization was achieved by combining the two approaches. In the present work, we show that the stabilization of the plasmid-encoded LacZ(+) phenotype can be explained solely by the effect on the growth rate ratio between cells containing modified and parental plasmids. By using modified stability experiments (where a single cell rather than a suspended colony was used to initiate growth), independent growth rate measurements, and a simple mathematical model, we can describe the kinetics of the loss of the LacZ(+) phenotype in terms of two variables, alpha and p (where alpha is the ratio of growth rates between modified and parental cells, and p is the probability of obtaining modified cells from parental cells). Under the conditions tested, the average values of alpha were 1.52 for cultures growing in complex medium, 1.28 for cultures growing in defined medium, and 1.18 for cultures containing the modified plasmid pYS1 growing in complex medium. The calculated p values ranged between 10(-8) and 10(-10) under all conditions. Plasmid (pYS137) was used to directly estimate plasmid deletion rates in B. subtilis and it showed a rate between 5 x 10(-8) and 1.1 x 10(-9) deletions/cell/generation. In contrast to B. subtilis, there were no detectable differences in growth rates between Escherichia coli strains harboring plasmid pCEDS and plasmid-free cells. These results explain the observed stability of pCEDS in E. coli cultures and indicate that readily detected instability in B. subtilis cultures can be the result of rare deletion events.     

双歧杆菌质粒聚合酶基因表达系统的构建及其鉴定

目的构建可以在双歧杆菌表达外源性基因的系统。方法PCR扩增双歧杆菌质粒聚合酶基因（BPP）并连接至质粒pBS—T以形成重组质粒pBS—BPP。PCR扩增双歧杆菌内源性阿拉伯糖苷酶的启动子及分泌性信号肽DNA序列（ara）并连接至质粒pBAD—A以形成重组质粒pBAD—ara,然后将增强绿色荧光蛋白（eGFP）基因连接至质粒pBAD—ara以形成重组质粒pBAD—ara—GFP。采用基因重组技术重组含有ara、BPP、eGFP基因序列并可将外源性基因分泌表达于菌体外及锚定表达于细胞壁的质粒pBS—BPP—ara—GFP,激光共聚焦显微镜下观察含有pBS—BPP—ara—GFP质粒及对照质粒的E．coli,验证eGFP定位表达情况。结果所构建的表达系统可以在E．coli中表达eGFP基因。结论通过基因重组方法成功构建了双歧杆菌表达系统,其可将外源基因分泌表达于菌体外。